Isolation and Identification of Secondary Metabolites from the Aerial Parts of Stachys lavandulifolia Vahl.

Stachys lavandulifolia Vahl is an herbaceous wild plant native to Iran which is traditionally used in Iranian folk medicine as a mild sedative tea for reducing anxiety and for treatment of gastrointestinal disorders. Our previous study on ethyl acetate extract of S. lavandulifolia proved anti-anxiolytic activity and so the present study was designed to determine chemical components of this biologically active fraction. The extract was prepared using maceration method. Column chromatography and medium pressure liquid chromatography (MPLC) was used respectively to separate the fractions. Finally, some evaluated fractions were used for high pressure liquid (HPLC) and peak shaving recycle technique to achieve more purification. Separated compounds were determined using NMR analysis and mass spectroscopy. Six compounds have been isolated from ethylacetate extract of aerial parts of S. lavandulifolia including four flavonoids (apigenin, kumatakenin, penduletin and 4', 7-dihyroxy- 3, 5, 6-trimethoxy flavon), a labdan diterpenoid (labda-13-en-8, 15-diol), and an iridoid.


Introduction
The genus Stachys is one of the largest genera in the flowering plant family of Lamiaceae with about three hundred species distributed in Europe, Asia, Africa, Australasia, and North America(1).
Stachys lavandulifolia Vahl is an herbaceous wild plant native to Iran (2) which is used in Iranian folk medicine as a mild sedative tea for reducing anxiety and for treatment of gastrointestinal disorders (3).
Previous study on hydroalcoholic, polyphenolic, and boiled extracts of S. lavandifolia demonstrated their analgesic effect on animal models (4). In another study, antimicrobial and antioxidant activities of the essential oil of S. lavandulifolia were observed (5). Our previous study on S. lavandulifolia hydroalcoholic extract by the elevated plusmaze (EPM) model of anxiety in mice showed anxiolytic effect at therapeutically acceptable doses (6). Further study on petroleum ether, ethyl acetate, butanol and aqueous fractions on spontaneous motor activity and elevated plus-maze behavior in mice showed interesting anxiolytic properties for ethyl acetate fraction (7) and thus, we decided to investigate the main chemical constituents responsible for anxiolytic effects of ethyl acetate extract of S. lavandulifolia.
Plant material S. lavandulifulia was collected from Shahrekord city in Chaharmahal va Bakhtiari Province in the west of Iran. The plant was identified by the Department of Biology, Faculty of Science, University of Isfahan and also a voucher specimen (No. 1113) was deposited in the herbarium of the Isfahan Faculty of Pharmacy, Isfahan University of Medical Sciences, Isfahan, Iran.

Results and discussion
The 13 C NMR and 1 H-NMR spectra of compound 1 indicated signals of three singlet methyl groups, two methylene, an oximethine, two quaternary carbons which one of them was oxygenated, a tri-substituted olefin bond and a lactone carbonyl with EIMS m/z 196 which resembled with those of a C11 iridoid named loliolide (10). The iridoids and secoiridoids form a large group of plant constituents that are found usually. They are mostly 4, 7-dimethylcyclopentapyran. But in the case of loliolide (C11 iridoid) and its related structures like actinidiolide and aeginetolide their biosynthesis pathway is different from usual C-10 iridoids and secoiridoids. They are generated in variety of plants from photo-oxygenation and degredation of carotenoids (11-12).
Compound 2 was obtained as a white powder. 13 C-NMR data in addition to positive ESIMS at m/z 331 [M+Na] + corresponded to the formula C 20 H 36 O 2 . 1 H NMR spectrum showed signals attributed to five singlet methyls δ H 1.70, 1.14, 0.90, 0.86, and 0.84. Further signals in 1 H NMR spectrum at δ H 5.38 (t, 1H, J=6.8Hz, H-14) and 4.09 (d, 2H, J=6.8Hz, H-15) indicated the presence of a tri-substituted olefin bond and an external oxymethylen group. The 13 C-NMR and DEPT spectra demonstrated twenty carbons comprised of five methyls, eight aliphatic methylens (one oxygenated), two aliphatic methines, two aliphatic quternary carbons (one oxygenated), one olefinic methine, and one quaternery olefinic carbon. Three degrees of unsaturation, and one olefinic bond suggested two rings in the structure. Comparing these characteristic signals with compound 6 isolated from Leonurus heterophyllus by Hung and coworkers (14) and compound 8 isolated from Leonurus heterophyllus by Giang and coworkers (13) and using HMBC spectra determined structure of compound 2 as labda-13-ene-8, 15diol.
Compound 3 was isolated as a yellowish powder with orange fluorescence reaction to Natural Product reagent (Diphenylboric acid 2-aminoethyl ester 1%). Molecular formula was determined by NMR data and negative EIMS m/z 344 [M] as C 18 H 16 O 7 . Eleven degrees of unsaturation, one carbonyl carbon, and seven olefin bands indicated three rings in the structure. The 1 H-NMR spectrum showed a singlet olefin proton at δ 6.79 (s, 1H) described to H-8. Orthocoupled proton signals( each two protons) at δ 8.05 (d, 2H, J=8.8Hz) and 6.95 (d, 2H, J=8.8Hz) were corresponded to H-2',6ʹ and H-3ʹ, 5ʹ. Three singlet methoxy signals at δ H 3.99 (δ C 61.1), 3.86 (δ C 60.6), 3.82 (δ C 57.0) were located on C-3, C-6 and C-7, respectively based on long range HMBC correlation and cleavage pattern of compound in EI-Mass by retro-Diels-Alder mechanism including A (m/z 197), B (m/z 149) and C (m/z 93) ions. All of these data defined the structure of 3 as 3, 6, 7-trimethoxy-4ʹ,5dihydroxyflavone named as penduletin which was in agreement with literature data (15). Figure 2 shows the fragmentation pattern of this compound by cleavage of ring C via retro-Diels-Alder mechanism.
Compound 4 assigned the molecular formula of C17H14O6 based on negative pseudomolecular ion [M -H]at m/z 313 and 13 C-NMR (BB and DEPT). 1 H & 13 C-NMR spectral data cleared that compound 4 and 3 have similar structures but differed in lacking methoxy group at C-6. Comparison of the spectral data with literature data determined the structure as 7,3-dimethoxy-4ʹ,5-dihydroxyflavone named as Kumatakenin (16). Similarly, NMR spectral data cleared that compounds 5 and 3 resembled each other, except for the 7-O-hydroxyl instead of 7-O-methoxyl group which was confirmed by pseudo-molecular ESI Mass ion [M -H]at m/z 329. It was defined as 6, 3-dimethoxy-4ʹ, 5-dihydroxyflavone (17). Compound 6 was proved to be 4ʹ, 5, 7-trihydroxyflavone named as apigenin (17).
Many species of the genus Stachys have been investigated and different kinds of secondary metabolites have been isolated, mainly flavonoids, iridoids and terpenoids from the aerial parts and the roots (18). In this investigation, an iridioid, a labdane type diterpene, three methoxylated flavonol derivatives, and a flavone were isolated.
Previous phytochemical studies showed presence of several types of flavonoids in Stachys genus. Some Stachys species are rich in flavonoids, for example 24 flavonoids were identified in S. aegyptica and 9 in S. ionica (19,20). However in this study, three methoxylated flavonol derivatives, and a flavone were isolated from S.lavandulifolia. Compound 3 with trivial name of kumatakenin, previously was isolated from Ballota hirsuta, Eupatorium illitum, Achillea kotschya, and Baccharis petiolata. The flavonoids from Eupatorium illitum have been presented antiproliferative activity (22). Compound 4 with trivial name of penduletin, was previously isolated from Achillea nobilis, Isocoma tenuisecta and Betola nigra (21). Pendulentin has showed antiproliferative property (23) and strong activity against enterovirus (24). Compound 5, was previously reported from Achillea kotschya, Achillea nobilis, and some other genera of Asteraceae and apigenin is also determind in many Stachys species 21). The diterpenoids isolated from different species of Stachys are structurally different with labdane, kauran, clerodane, and abietan skeletone. Kauran diterpenoids are present in S. silvatica, S. lanata, and S. sprumeri and also S. recta, neo-clerodan derivatives are found in S. annua, S. rosea, and S. recta. An abietan derivative in S. officinalise and diterpenoids with labdane skeleton were also obtained from S. mucronata, S. plumasa, and S. menthifolia (18, 25). The labdane diterpenoid which is isolated in this study was also previously reported from S.menthifolia, Leonurus heterophyllus, and Cistus creticus (11, 18).

References
including A (m/z 197), B (m/z 149) and C (m/z 93) ions. All of these data defined the structure of 3 as 3, 6, 7-trimethoxy-4',5-dihydroxyflavone named as penduletin which was in agreement with literature data (15). Figure 2 shows the fragmentation pattern of this compound by cleavage of ring C via retro-Diels-Alder mechanism. Comparison of the spectral data with literature data determined the structure as 7,3-dimethoxy-